Human intestines contain various microorganisms propagating therein, composing normal intestinal flora. Composition and activity of such intestinal flora may widely influence nutrition, bio-functionality, drug efficiency, carcinogenesis, aging, immune responses, resistance to infection, and the body's responses to other stresses as well as heath of animal and human, in positive manners. The intestinal flora comprises more than 500 different species. A relatively small number of bacteria reside in the stomach and the upper part of the small intestine, while increasing in number in the large intestine. As described above, the intestinal flora offers positive effects on human health. It is known that Bifidobacterium sp. in normal intestinal flora is of high importance. Such a fact can be inferred by observing that Bifidobacterium sp. inhabits the large intestine of humans, throughout their entire lives. Also, it was revealed that breast-fed infants have a larger number of Bifidobacterium sp. than bottle-fed infants, leading to the breast-fed infants having a lower occurrence of diarrhea. However, it is known that the number of Bifidobacterium sp. is rapidly decreased with aging.
Distribution of the normal intestinal flora in humans changes depending on diverse factors such as age, race, living environments, diet, etc. The diet especially may have big effects on the normal intestinal flora. Reportedly, people living a healthy long life or people with low incidence of adult diseases have a large number of intestinal Lactobacillus, compared to other people, For this reason, the importance of selecting foods to elevate well-balanced distribution of the intestinal flora is rising. With an aim of obtaining well-balanced distribution of the intestinal flora by ingesting Lactobacillus strains, active studies for development of food products prepared by adding Lactobacillus to conventional dairy products, including yogurts, are underway.
Thus, there have been many attempts to use Lactobacillus in producing health foods and medicaments, as interest in personal health is rising. Actually, various foods and medicaments comprising Lactobacillus have been developed. The Lactobacillus strains, which are currently used in Korea, are all developed in foreign countries, so there may be differences in their characteristics from strains residing in the intestines of Korean people. It is expected that the Lactobacillus strains residing in the intestines of only Korean people may exhibit higher physiological activities, due to their greater adaptability to the Korean people, than those of any other Lactobacillus strains developed up to now. Accordingly, attempts for development of novel foods and medicaments comprising Lactobacillus strains which are fit to Korean people, by isolating useful Korean-type Lactobacillus strains from Korean people, have been made.
Bifidobacterium sp. is Gram-positive and Y-shaped or V-shaped. It is nonmotile, anaerobic, nonsporing, and fermenting. Bifidobacterium sp. is traditionally categorized as lactic acid bacteria. Fermentation by Bifidobacterium sp. leads to production of acetate and lactate as main products, at a ratio of 3:2. The GC content of its DNA is 55 to 64%. Bifidobacterium sp. is known to be host-specific. In humans, B. bifidum, B. longum, B. breve, B. infantis, and B. adolescentis are found. The above five species have been used as probiotics. Probiotics refers to microorganisms as nutrients which exhibit beneficial effects on their hosts by maintaining desirable balance of intestinal microbes and enhancing physiological characteristics thereof (Fuller, R., Journal of Applied Bacteriology, 66: 365-378, 1989).
To date, many vector systems derived from Lactobacillus sp. and Lactococcus sp., both being categorized as lactic acid bacteria, have been developed. It is expected that commercialization of such vector systems based on technologies of gene modification is realizable. Development of vector systems derived from Bifidobacterium sp., which is better suited to the intestines, is still in initial stages. Plasmids derived from Bifidobacterium strains so far studied are described below.
In the early research, it was believed that only B. longum among bacteria isolated from humans carries a plasmid. The plasmid isolated from the B. longum was ligated with a vector for transformation, derived from E. coli thus developing a shuttle vector. It was reported that plasmids exist in B. globosum, B. asteroides, and B. indicum. The plasmids are grouped according to their genetic homology, as determined by electrophoresis patterns or Southern hybridization. It was also reported that another strain, B. breve has a plasmid (Sgorbati, B. et al, Microbiologica., 6: 169-173,1983; Tannock, G. W. et al., J. Clin. Microbiol., 28: 1225-1228, 1990).
Vectors using plasmids of Bifidobacterium strains were rarely reported until the 1990s. B. longum B2577 harbors a plasmid pMB1 1.9 kb long. The pMB1 was cloned into an E. coli-derived vector, and a cleavage map thereof was generated. In 1994, a shuttle vector pRM2 that is replicated in E. coli and B. longum was developed using pMB1 as a basic vector, and transformation of B. longum via electroporation was achieved. Recently, the entire nucleotide sequence of pMB1 was determined (Mateuzzi, D., et al., Letters in Applied Microbiology, 11:220-223, 1990; Missichi, R., et al, Plasmid,32:208-211, 1994; Argnami, A. et al., Microbiology, 142:109-114, 1996). The Bifidobacterium sp. has a thick multi-layered cell wall. The cell wall contains diverse components such as peptidoglycan, polysaccharide, lipoteichoic acid and protein. Such characteristics of the cell wall contribute to its role as a barrier against intake of foreign DNA molecules. For the purpose of transforming Bifidobacterium sp., although various attempts have been made in terms of academic or industrial applications, only few successes have been achieved. There was a report in which B. longum was employed to construct a transformant through low efficiency electroporation (Missich, R et al., Plasmid,32:208-211, 1994).
Using the plasmid pMB1 isolated above, a shuttle vector pNC7 was constructed, and a transformation method for application in various species of Bifidobacterium sp. was developed. The transformation efficiency varied between 1.0×101 to 1.2×105 CFU/μg DNA, according to species. According to the method, Actilight®P was used as a glucose source in a medium, and the bacteria in log-growth phase were collected, preparing competent cells. The conditions for electroporation were set to 12.5 kV/cm, 100 Ω, 25 μF to perform transformation. In Japan, a shuttle vector which is replicated in E. coli and Bifidobacterium sp. was developed using a 3.6 kb plasmid (pTB6) isolated from B. longum (see Table 1) (Matsummura, H. et al., Biosci. biotech. Biochem., 61(7):1211-1212, 1997).
Meanwhile, Korean researchers first isolated plasmids from Bifidobacterium strains in 1994, and the plasmids were divided into several homology groups based on results of Southern hybridization (Hyun-Seo Jung et al., presented at the Spring Conference of the Korean Society of Food Science and Technology, 1994; Joo-Hun Lee et al., presented at the Spring Conference of the Korean Society of Food Science and Technology, 1997). Among those strains, two strains exhibited relatively strong resistance to erythromycin and tetracycline. Such resistance of the strains was considerably reduced upon loss of those plasmids. Another strain B. longum KJ harbors two distinct plasmids pKJ36 and pKJ50 (see Table 1). Each of these plasmids was cloned into an E. coli-derived vector for transformation, thereby generating cleavage maps thereof. Those entire nucleotide sequences were found, compared and analyzed (Myoung-Soo, Park et al., presented at the Fall Conference of the Korean Society for Applied Microbiology, 1995; 1996; 1997). Results showed that the two plasmids have very similar characteristics, and each contains an ORF which shares significant amino acid homology with Rep and Mob proteins expressed from certain plasmids in Gram-positive and Gram-negative bacteria. Each ORF was expressed at translation/transcription levels. Based on those results, shuttle vectors pBKJ50F, pBKJ50R and pBRepA which replicate in E. coli and Bifidobacterium sp. were constructed to use in transforming Bifidobacterium sp.
As described above, all the vectors developed by using the Bifidobacterium—derived plasmids contain a foreign antibiotic resistance gene as a selection marker. It is a shortcoming that they cannot be applied directly for foods. Therefore, there is a need for development of a food-grade vector which can be used for food in a safe manner. Development for new shuttle vectors is ongoing in such manners that antibiotic resistance genes are removed for replacement with a food-grade selection marker, and promoter/operator sequences are inserted to regulate foreign gene expression. Table 1 shows plasmids in Bifidobacterium sp. and shuttle vectors derived therefrom.
TABLE 1Plasmid (kb in size)Host cellVectorSelection markerpMB1(1.9)B. longumpRM2SpectinomycinaAmpicillinbpNC7ChloramphenicolaAmpicillinbpKJ36(3.6)B. longum KJpEKJ36ChloramphenicolaAmpicillinbpKJ50(5.0)B. longum KJpBKJ50FChloramphenicola,bpBKJ50RChloramphenicola,bpBRepApNBb1(5.6)B. breveNotdevelopedpTB6(3.6)B. longumpBLES100StreptomycinaAmpicillinbNote: aselection marker expressed in Bifidobacterium sp. bselection marker expressed in E. coli 
In view of the above problems, the inventors have made an effort to develop a shuttle vector which is replicated in both E. coli and Bifidobacterium sp. and express a target gene, thereby capable of being used as a food additive and for preparing oral vaccines without the need for purification upon expression of the target gene in Bifidobacterium sp., and a promoter which directs a strong expression of the target gene in Bifidobacterium sp.